Few studies have examined the relationship between teleworking practices and presenteeism. This study determined the association between teleworking practices and presenteeism among teleworkers in Japan. A cross-sectional online survey was administered to 2687 teleworkers from five companies in Japan, collecting data on demographic variables, teleworking practices, frequency and duration of teleworking, presenteeism, and various lifestyle- and health-related factors. A logistic regression analysis was performed. Teleworkers with full-time employment and less teleworking experience exhibited higher presenteeism rates. Key practices negatively associated with presenteeism included creating a dedicated workspace, chatting with colleagues, and setting daily work goals. Gender differences were significant: for men, additional practices, such as determining their work hours, were beneficial; while for women, chatting with colleagues was particularly important. A sensitivity analysis indicated that specific teleworking practices can mitigate presenteeism. Certain teleworking practices, such as creating a workspace, chatting with colleagues, and setting work goals, were associated with lower presenteeism among teleworkers. These findings highlight the need for organizations to support telework ergonomics, promote social interaction, and encourage goal setting to enhance teleworker productivity and health. Training for employees and supervisors to raise awareness of their own and their subordinates’ health while teleworking is advised.